2.3　Flow cytometry

Apoptotic cells were quantified by flow cytometry［14］using the Propidium Iodide(PI)-Annexin V/fluorescein isothiocyanate(FITC)Apoptosis Detection Kit(BD Pharmingen，5043815).A total volume of 2mL of MIN-6 suspension cells at 2×105 cells/mL were seeded in each well of a 6-well microtitre plate and incubated at 37℃for 24h.The cells were grown in different media for each treatment group：basic medium basic medium with 33.3mmol/L glucose；basic medium with 1μL/mL XKP basic medium with 33.3mmol/L glucose and 1μL/mL XKP mixture；basic medium with 33.3mmol/L glucose，1μL/mL XKPand 2mmol/L 3-MA.After 36h at 37℃，the cells were washed with 1mL of phosphate buffered saline(PBS)solution and trypsinized with 350μL of trypsin-EDTA to be collected into the same centrifuge tube for each well and was then centrifuged at 1000rpm for 10min.The supernatant was discarded；the precipitant was washed with 1mL of PBS solution and centrifuged at 1000 rpm for 10min.We repeated the wash process twice.The precipitant was resuspended in 100μL annexin binding buffer(1×)filtered by 70μm Cell Strainers(Biologix，15-1070)，and then transferred to a new tube.In each tube，5μL of Annexin V-FITC was added and the tube was placed in ice for 10min.Then 5μL of PI was added and tubes were incubated for five more minutes in ice.Another 400μL annexin binding buffer(1×)was added to each tube and the solution was analysed by flow cytometry(BD FASCCanto II)with Cell Quest software(BD Sciences，America).(https://www.daowen.com)